Colony formation assay PANC 1 cells were seeded in 6 well plates, and then treated or untreated with radiation and AZD 8055, alone or in combination. The medium was re placed with fresh medium containing selleck MEK162 the reagent and radiation treatment every three days. After 10 days treat ment, the medium was removed and cell colonies were stained with crystal violet. Pic tures were taken using a digital camera to record the re sult as described. To evaluate the colony formation ability of irradiation resistant cells, PANC 1 irradiation resistant cell line was firstly generated by plating PANC 1 cells in 100 mm culture dishes and ir radiating with 2 Gy X ray every three days over a period of 5 months, for a total dose of 100 Gy, and then colony formation assay was used as above mentioned.

Transfection PANC 1 cells were suspended in DMEM supplemented with 10% FBS and seed in 6 well plates and transfected Inhibitors,Modulators,Libraries with miR 99b precursor or inhibitor with Lipofectamine 2000 according to the manufacturers instruction. After 48 h of transfection, cells were Inhibitors,Modulators,Libraries treated by radiation at 5 Gy, then harvested and lysed for Western blot assay. Apoptosis analysis Annexin V PI Apoptosis Detection kit was used for quantification of apoptosis. Cells were seeded in 6 well plates in the absence or presence of AZD8055, then radiation was applied 4 h later. After cultured for 24 h, 0. 5 1 106 cells were collected into each tube and gently washed with PBS. Cell pellets were suspended in 1 binding buffer and stained with Annexin V and PI. After incubated for 15 min at RT in the dark, the apoptosis analysis was carried out using a FACScan and analyzed using FlowJo software.

Cell cycle analysis Inhibitors,Modulators,Libraries Cells were synchronized by growing in serum free medium for 48 h and then released Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries into the cell cycle by adding 10% FBS to the medium. The cells were treated with radi ation in the absence or presence of AZD8055 for 24 h, harvested, fixed with 70% ethanol, and stained with PI. Data were acquired using flow cytometry and ana lyzed using FlowJo software. Pancreatic cancer xenografts and treatments Animal experiments were careful to follow the protocols approved by Jilin University and the Fourth Military Medical University Institutional Animal Care and Use Committees. PANC 1 cells were resuspended in HBSS and injected subcutaneously into the flank region of 6 week old female athymic mice.

The tumors were allowed to grow selleck inhibitor to average volume of 200 mm3 prior to initiation of therapy as described. Then mice were assigned randomly to four groups as following vehicle control. 8 Gy fractionated radiotherapy. the radiation was performed using the same X ray machine with a different filter, at a dose rate of 1 Gy min. AZD8055, AZD8055 was dissolved in DMSO and administered by oral gavage. Combination of AZD8055 and 8 Gy fractionated radiotherapy. Tumor volumes were measured with a caliper every other day and calcu lated based on the formula V 4 3 �� 2.